As a means for transporting a medicament specifically to a pathological lesion, methods of encapsulating a medicament in liposomes have been proposed. In particular, in the field of therapeutic treatments of malignant tumors, many reports have been made as for effectiveness of liposomes encapsulating an antitumor agent. Further, a multifunctional envelope-type nano device (MEND: henceforth sometimes abbreviated as “MEND” in the specification, see, for example, Drug Delivery System, 22-2, pp. 115-122, 2007 and the like) has been proposed. This structure can be used as a drug delivery system for delivering a gene or the like selectively into particular cells, and is known to be useful for, for example, gene therapy of tumors and the like.
Variety of methods have been proposed for modifying the surface of a lipid membrane structure with a functional molecule, as means for delivering an objective substance such as medicaments, nucleic acids, peptides, polypeptides, and saccharides to specific parts such as target organs and tumor tissues using a lipid membrane structure. When a lipid membrane structure encapsulating a medicament such as antitumor agent reaches a target cell, the structure is taken up into the cell by endocytosis and encapsulated in the endosome. Then, the structure releases the encapsulated medicament into the cytoplasm due to hydrolytic action of an enzyme in the lysosome or the like. In order to enhance the release of medicament from a liposome taken up into the endosome, a liposome has been proposed of which surface is modified with a peptide, GALA (Biochemistry, 26, pp. 2964-2972, 1987 for the peptide; Biochemistry, 43, pp. 5618-5623, 2004 for the liposome) and MEND (Japanese Patent Unexamined Publication (KOKAI) No. 2006-28030).
Further, as means for localization of a lipid membrane structure encapsulating an objective substance such as nucleic acid into the nucleus of a target cell, there have been proposed, for example, a liposome of which outer surface is modified with octaarginine (International Patent Publication WO2005/32593; Journal of Controlled Release, 98, pp. 317-323, 2004), a bilamellar liposome having a lipid membrane modified with a nucleus permeable peptide (International Patent Publication WO2006/101201), and a liposome of which surface is modified with a monosaccharide such as galactose and mannose (International Patent Publication WO2007/102481). It has been reported that a multilamellar lipid membrane structure (T-MEND) modified with a monosaccharide has fusability with a lipid membrane and a nuclear membrane, and is capable of improving gene expression efficiency as an experimental result in vitro. It has further been reported that a lipid membrane structure modified with the KALA peptide (Biochemistry, 36, pp. 3008-3017, 1997) can efficiently deliver such a substance as a nucleic acid into a nucleus of a cell (WO2011/132713).
Dendritic cells are antigen-presenting cells responsible for the central part of immunological response, therefore they constitute one type of important target cells of cancer immunotherapies, and they are also used for immunocyte therapy (dendritic cell therapy), in which dendritic cells are extracted from a cancer patient, subjected to antigen introduction or activation outside the body, and then administered again to the patient. Since an immunosuppressive factor has been discovered in dendritic cells in recent years, they attract attention also as a target of siRNA medicaments, and it is expected that combination of them with the dendritic cell therapy shall realize more potent cancer immunity induction.
Concerning introduction of siRNA into nuclei of dendritic cells, there are former reports that an immunosuppressive factor was knocked down by using a lentivirus vector that expresses shRNA (Nat. Biotechnol., 2004; Nat. Med., 2008). However, there are almost no reports concerning introduction of siRNA into dendritic cells using an artificial delivery system. Although use of a virus vector can achieve efficient knockdown of a target gene, it has a problem from a viewpoint of safety.
As an artificial delivery system for introduction of siRNA, R8/GALA-D-MEND (D-MEND) has been reported (J. Control. Release, 143, pp. 311-317, 2010). D-MEND is a nano-carrier consisting of MEND of which envelope membrane number is controlled by modification with an octarginine (R8) peptide as a cellular affinity device and a GALA peptide as an endosomal escaping device. D-MEND gives about 70% of knockdown even at such a low siRNA concentration as 12 nM in HeLa cells, which are generally used cancer cells, and the activity thereof is twice or more higher than that of Lipofectamine 2000 (LFN2000), which is widely used as a general introduction reagent.
However, in the case of transfection with D-MEND into dendritic cells induced from mouse bone marrow cells, use of a high siRNA concentration (80 to 120 nM) is required in order to attain a knockdown efficiency of 70 to 80%, and it also suffers from a problem that the knockdown efficiency is limited to about 40% depending on the target factor of siRNA (Biol. Pharm. Bull., 34, pp. 1348-1351, 2011). As described above, use of a conventional artificial delivery system tends to markedly reduce the knockdown efficiency in dendritic cells, as compared with that obtainable in general cancer cells, and this fact hampers development of siRNA medicaments in the field of immunotherapy.